Drugs containing reduced of vitamin B2

ABSTRACT

The present invention provides medicines for preventing or treating infectious diseases, sepsis and/or septic shock, which exhibit excellent immunostimulating effects. More specifically, the invention provides medicines for immunostimulation and infection-protection and -treatment, and/or for preventing or treating sepsis and septic shock, which comprise reduced riboflavin and/or reduced riboflavin derivative or pharmacologically acceptable salt thereof. Also provided are methods for using them in the prevention and/or treatment of infectious disorders such as sepsis and septic shock.

PRIORITY INFORMATION

The present application claims the benefit under 35 U.S.C. § 371 ofInternational Application No.: PCT/JP02/02616 (published PCT applicationNo. WO 02/074313), filed 19 Mar. 2002, which claims priority to JapanesePatent Application No.: 2001-80578, filed 21 Mar. 2001, the entirecontents of each of these applications are hereby incorporated herein byreference.

BACKGROUND OF THE INVENTION

Antibiotics such as anti-bacterial agents and anti-fungal agents aretraditionally known to be effective for preventing or treating variousinfectious diseases and are widely used for clinical applications today.These antibiotics can be obtained by the extraction of products frombacteria or fungi or by chemical synthesis. However, when antibiotics ofthe same line are used repeatedly for a long period, the target bacteriamay acquire resistance against the antibiotics and the effect of theantibiotics may not be developed anymore. This emergence of so-calledresistant bacteria has become a serious social problem.

Thus, alternative approaches for protecting hosts via routes differentfrom those of antibiotics have been considered. One representativeexample involves the potentiation of the immune functions of a host toprotect the host from microbial infections. Examples of drugs useful forthis approach include immunopotentiators/infection defense medicines andmedicines for treating infections comprising riboflavin, which is knownwidely as vitamin B₂, or riboflavin derivatives such as riboflavinmononucleotides, and flavinadenine dinucleotides as described in thegazette of Japanese Kokai Publication Hei-5-201864 or corresponding U.S.Pat. Nos. 5,814,632 and 5,945,420.

Ascorbic acid, known as vitamin C, is also known to have the similareffect. It is described in JP-A 9-301861 that ascorbic acid derivativesare effective on bacterial shocks.

Furthermore, the gazette of Japanese Kokai Publication 2000-297046discloses that an extract from sweet potato has an effect of stimulatingimmune of a living body and of preventing infections.

A systemic inflammatory reaction against infections, namely sepsis;i.e., systemic inflammatory response syndrome (SIRS), is a state where amicroorganism such as a bacterium or a fungus developed in an infectedhost is proliferated in a large quantity in the living body and thecells of the microorganism or a metabolite thereof is transferred to theblood and circulated through the body. As this state is progresses, anexotoxin excreted from the microorganism cells or an endotoxin releasedupon the disruption of the microorganism cells, or a componentconstituting the microorganism cells is transferred to organs of thehost. In the initial stage, fever, malaise and chill occur. As thecondition progresses, multiple organ failure develops and symptoms suchas impaired consciousness, dyspnea or decreased blood pressure occur,causing a state of shock often followed by death.

Consequently, potent antibiotics are administered to kill microorganismsin the infected host in the initial stage of sepsis, and systemicmanagement such as reinfusion or artificial respiration is applied andsimultaneously hemodialysis, plasma exchange or administration of a drug(catecholamine) is performed in the advanced stages. However, theefficacy of these approaches is not always satisfactory.

Recently, medicines for preventing or treating toxin shocks comprisingvitamin B₂ have been reported in the gazette of Japanese KokaiPublication Hei-10-29941. In addition, the gazette of Japanese KokaiPublication 2000-178246 discloses cycloalkene derivatives for use in thetreatment of septic shock; the gazette of Japanese Kokai Publication2000-80046 discloses medicines for preventing or treating syndromes thatdevelop in the exacerbated state of sepsis; and the gazette of JapaneseKokai Publication 2000-302768 discloses hydrazone derivatives for use inthe treatment of endotoxin shock. Furthermore, the gazettes of JapaneseKokai Publication Hei-5-201864 and of Japanese Kokai PublicationHei-10-29941 disclose medicines for immunostimulation andinfection-protection and -treatment comprising riboflavin and/or ariboflavin derivative as active ingredient and medicines for preventingor treating toxin shocks, respectively. However, no description aboutreduced riboflavin is found in these patent publications.

Turning to infections from the malaria protozoa, Japanese patentpublication No. Hei-6-506212 discloses that riboflavin is effective forthe prevention and treatment of malarian diseases. Furthermore,Antimicrobial Agents and Chemotherapy 44(1), 88-96, 2000, reports that“malaria parasite proliferates by digesting hemoglobin in erythrocytesand oxidizing the hemoglobin to convert it to methemoglobin. Sinceriboflavin is able to reduce methemoglobin to hemoglobin, the treatmentwith riboflavin reduced the amount of hemoglobin in the body of malariaparasite, resulting in inhibition of proliferation of malaria parasite.”

However, the medicines for immunostimulation and infection-protectionand -treatment, the medicines for preventing or treating sepsis and themedicines for preventing or treating septic shock as mentioned above arenot always effective on all patients or animals and the efficacy ofthese medicines is not also satisfactory. Therefore, the development ofmedicines for immunostimulation and infection-protection and -treatment,and for preventing or treating sepsis and septic shock with betterefficacy is desired.

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS OF THE INVENTION

The inventors have completed thorough research in light of suchcircumstances, the results of which have unexpectedly revealed thatreduced vitamin B₂ has a more potent immunoregulating effect thanaforementioned vitamin B₂, which culminated in the present invention.Specifically, reduced vitamin B₂ has been found to allow a living bodyto maintain homeostasis by stimulating the immune system when the immunefunction of the living body is reduced by infections or the like, or byinhibiting excess immunostimulation such as shocks and inflammatoryreactions when such reactions are found in the living body.

The present invention also relates to medicines for immunostimulationand infection-protection and -treatment, and/or medicines for preventingor treating sepsis, which comprise reduced vitamin B₂ (i.e., reducedriboflavin) and/or a reduced riboflavin derivative or pharmacologicallyacceptable salts thereof as active ingredient. Medicines for preventingor treating sepsis may be medicines for preventing or treating septicshock, disseminate intravascular coagulation (DIC), multiple organfailure (MOF) and/or adult respiratory distress syndrome (ARDS). Themedicines may also be for preventing or treating toxin shock as theseptic shock.

The present invention provides a method for immunostimulation andinfection-protection, or for preventing or treating sepsis, septic shockor malaria, which comprises administering a pharmacologically effectiveamount of reduced riboflavin and/or a reduced riboflavin derivative or apharmacologically acceptable salt thereof to a subject.

The present invention also provides use of reduced riboflavin and/orreduced riboflavin derivatives or pharmacologically acceptable saltsthereof for the production of medicines for immunostimulation andinfection-protection and -treatment, and/or medicines for preventing ortreating sepsis, septic shock or malaria.

The present inventors have found that, among riboflavin derivatives,particularly reduced forms thereof have remarkably strongimmunostimulating and infection-protective/curative effects,sepsis-preventive/curative effects and septic shock-preventive/curativeeffects and/or toxin shock-preventive/curative effects, which culminatedin the present invention.

The present invention also provides medicines for preventing or treatingmalarian diseases comprising reduced riboflavin and/or a reducedriboflavin derivative or a pharmacologically acceptable salt thereof asactive ingredient. Since reduced forms of riboflavin and/or riboflavinderivatives, or pharmacologically acceptable salts thereof have a higherreductive effect than riboflavin and/or riboflavin derivatives orpharmacologically acceptable salts thereof, it is proposed that they caninhibit the growth of malaria parasites more effectively.

Medicines for immunostimulation and infection-protection or -treatment,for preventing or treating sepsis, for preventing or treating septicshock and/or for preventing or treating malaria according to the presentinvention include the cases where a precursor of reduced riboflavinand/or a reduced riboflavin derivative or pharmacologically acceptablesalt thereof (e.g., riboflavin and/or a riboflavin derivative orpharmacologically acceptable salt thereof) administered to a subject maybe converted (e.g., reduced) in vivo to reduced riboflavin and/or areduced riboflavin derivative or pharmacologically acceptable saltthereof, which exhibits an immunostimulating andinfection-protective/curative effect, a septic shock-preventive/curativeeffect or a malaria-protective/curative effect in the subject.

Reduced riboflavin and/or reduced riboflavin derivatives orpharmacologically acceptable salts thereof according to the presentinvention refers to a reduced form of vitamin B₂ and is not particularlylimited. Preferably the term encompasses reduced forms of riboflavin,flavin mononucleotide (riboflavin phosphate), flavin adeninedinucleotide and riboflavin tetrabutylate, and pharmacologicallyacceptable salts thereof. Reduced riboflavin may be, for example,leucoflavin or monohydroflavin or pharmacologically acceptable saltsthereof. Reduced riboflavin derivatives may be, for example, leucoflavinmononucleotide (FMN-H₂), leucoflavin adenine dinucleotide (FAD-H₂) orpharmacologically acceptable salts thereof. The present inventionencompasses pharmacologically acceptable salts, hydrates and the like ofreduced riboflavin.

Reduced riboflavin and/or reduced riboflavin derivatives orpharmacologically acceptable salts thereof according to the presentinvention can be produced readily by the addition of a conventionalreducing agent such as a hydrosulfite or tin chloride. For example, theycan be produced by the method described in the Japanese Pharmacopoeia,12^(th) revision (Hirokawa-Shoten, 1991), C-2278 (the article of“riboflavin”), “riboflavin can be reduced by reduction or catalyticreduction with a hydrosulfite or tin chloride to a leuco-typedihydroflavin (also referred to as “leucoflavin”) through asemiquinone-type monohydroflavin”. For example, 120 mg of riboflavinsodium phosphate (FMN-Na) can be dissolved in 400 ml of physiologicalsaline and then mixed with 4.8 g of sodium hydrosulfite to produceleucoflavin sodium phosphate (FMN-Na-H₂). Of course, the method forproducing reduced riboflavin and/or reduced riboflavin derivatives orpharmacologically acceptable salts thereof of the present invention isnot limited to this method.

Riboflavin, a monohydroflavin and a dihydroflavin (leucoflavin) can beproduced mutually by a oxidation/reduction reversible reaction.Riboflavin and/or riboflavin derivatives or pharmacologically acceptablesalts thereof are surmised to be converted into reduced riboflavinand/or reduced riboflavin derivatives or pharmacologically acceptablesalts thereof via in vivo reduction. Therefore, in medical applicationsof reduced riboflavin and/or reduced riboflavin derivatives orpharmacologically acceptable salts thereof according to the presentinvention, riboflavin and/or a riboflavin derivative orpharmacologically acceptable salt thereof (e.g., riboflavin sodiumphosphate) may be administered to a subject to induce the production ofreduced riboflavin and/or a reduced riboflavin derivative orpharmacologically acceptable salt thereof (e.g., leucoflavin sodiumphosphate, monohydroflavin sodium phosphate) in the patient subject viain vivo metabolism, which exhibits the pharmacological activities of thepresent invention. This case is also encompassed in the presentinvention.

Accordingly, the present invention provides a method for the preventionand treatment of a target disease described in the present invention,which comprises administering riboflavin and/or a riboflavin derivativeor pharmacologically acceptable salt thereof to produce reducedriboflavin and/or a reduced riboflavin derivative or a pharmacologicallyacceptable salt in vivo. Preferably, the present invention provides amethod for the prevention and treatment of a target disease described inthe present invention, which comprises administering riboflavin sodiumphosphate to produce leucoflavin sodium phosphate and/or monohydroflavinsodium phosphate in vivo.

The present invention also provides a method for immunostimulation andinfection-protection, or preventing or treating sepsis, septic shock ormalaria by the action of reduced riboflavin and/or a reduced riboflavinderivative or pharmacologically acceptable salt thereof which isproduced in vivo, which comprises administering a pharmacologicallyeffective amount of riboflavin and/or a riboflavin derivative orpharmacologically acceptable salt thereof to a subject.

The present invention also provides medicines for immunostimulation andinfection-protection and -treatment, and/or for preventing or treatingsepsis, septic shock or malaria comprising, as active ingredient,reduced riboflavin and/or a reduced riboflavin derivative orpharmacologically acceptable salt thereof which is produced in vivo byadministering riboflavin and/or a riboflavin derivative orpharmacologically acceptable salt thereof.

The present invention also provides use of riboflavin and/or ariboflavin derivative or pharmacologically acceptable salt thereof forthe production of medicines for immunostimulation andinfection-protection, or for preventing or treating sepsis, septic shockor malaria by the action of reduced riboflavin and/or a reducedriboflavin derivative or pharmacologically acceptable salt thereof whichis produced in vivo by administering riboflavin and/or a riboflavinderivative or pharmacologically acceptable salt thereof.

Subjects to be administered with medicines of the present inventioninclude humans and animals.

The term “animal” as used herein refers to an industrial animal, acompanion animal or a laboratory animal. The industrial animal is ananimal whose breeding is required for industrial purposes, includinglivestock animals such as cattle, horse, pig, goat or sheep; farm fowlssuch as chicken, duck, quail, turkey or ostrich; fishes such asyellowtail, young yellowtail, red sea beam, horse mackerel, carp,rainbow trout or eel. The companion animal refers to a so-called pet,including dog, cat, marmoset, small birds, hamster and gold fish. Thelaboratory animal refers to an animal used for studies in the fields ofmedicine, biology, agriculture, pharmacy and the like, including rat,guinea pig, beagle, miniature pig, rhesus monkey and cynomolgus monkey.

For the administration to a human or animal which has not been infectedyet, which has not developed sepsis, septic shock or toxic shock, orwhich has been recovered from any one of these diseases, the medicinemay be used as a preventive medicine. For the administration to a humanor animal after infection or during the development of any one of thesediseases, the medicine may be used as a therapeutic medicine.

The term “sepsis” as used herein refers to a state where microbialinfection is developed systemically, or a state where a subject isinfected with a microorganism topically or systemically and an exotoxinexcreted from the microorganism, an endotoxin excreted upon thedisruption of the microorganism or a component constituting themicroorganism is spread systemically.

The term “septic shock” as used herein refers to a state wheredysfunction of at least one organ (e.g., heart, lung, liver, kidney,spleen, brain and spinal cord) occurs as the result of the systemicspread of the above-mentioned exotoxin, endotoxin or themicroorganism-constituting component, or a state where a symptom such asweakness, vertigo, dysstasia, decreased blood pressure, fall of bodytemperature, arrhythmia, ventricular fibrillation, dyspnea, fall of bodytemperature, convulsions, clouding of consciousness and unconsciousnessoccurs as the result of the dysfunction of the organs.

Medicines according to the present invention may be administered by anyroute depending on the purposes of the administration, symptoms or thelike, and may be administered by an intravenous, intramuscular,intraperitoneal, subcutaneous, transdermal or oral route, or in the formof an eye drop. Preferably, the medicines are administeredintravenously, intramuscularly or subcutaneously, particularlypreferably intravenously.

The dosage may vary depending on the purposes of administration, typesof the diseases to be treated and state of the symptoms. For intravenousadministration, the dosage may be 0.1 to 50 mg/kg, preferably 0.3 to 20mg/kg, more preferably 0.3 to 2 mg/kg. For intramuscular administration,the dosage may be 0.1 to 50 mg/kg, preferably 3 to 20 mg/kg. For oraladministration, the dosage may be 1 to 100 mg/kg, preferably 10 to 500mg/kg, more preferably 30 to 200 mg/kg.

Medicines according to the present invention may be administered withoutbeing processed or in the form of an injection solution (forintravenous, intramuscular, subcutaneous or intraperitonealadministration), an oral preparation (tablets, granules, powder,capsules), a transdermal preparation or an eye drop which may beprepared by mixing with any conventional pharmaceutical additives in aconventional manner. The medicine may also be blended in a food, a feed,drink water or the like.

Injectable preparations may be prepared by conventional methods, byadding, as needed, pH adjusting agents, buffering agents, emulsifyingagents, solubilizers, antioxidants, preservatives, isotonic agents orthe like to reduced riboflavin and/or reduced riboflavin derivatives, orpharmacologically acceptable salts thereof. The injectable preparationmay be freeze-dried to produce a lyophilized preparation of theextemporaneous type (i.e., the type of dissolving at the time of use).These injectable preparations may be administered intravenously,subcutaneously, intramuscularly or the like.

pH Adjusting agents and buffering agents include organic or inorganicacids and/or a salts thereof, sodium hydroxide and meglumine.Emulsifying agents include methylcellulose, polysorbate 80,hydroxyethylcellulose, gum arabica, sodium carboxymethylcellulose andpolyoxyethylene sorbitan monolaurate. Solubilizers includepolyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinic acidamide and polyoxyethylene sorbitan monolaurate. Antioxidants includeascorbic acid, α-tocopherol, ethoxyquin, dibutyl hydroxytoluene anddibutyl hydroxyanisole. Preservatives include methyl paraoxybenzoate,ethyl paraoxybenzoate and sorbic acid. However, they are not limitedthereto.

Oral solid preparations may be prepared in the form of tablets, coatedtablets, granules, powder or capsules by conventional methods, by addingas needed excipients, binders, disintegrating agents, lubricants,coloring agents, flavoring agents, anti-oxidants, solubilizing agents orthe like to reduced riboflavin and/or reduced riboflavin derivatives, orpharmacologically acceptable salts thereof.

Excipients include, but are not limited to, starch, corn starch,dextrin, glucose, lactose, saccharose, sugar alcohol, hydrogenated oil,mannitol, microcrystalline cellulose, anhydrous silicon, calciumsilicate and dibasic calcium phosphate. Binders include, but are notlimited to, polyvinyl pyrrolidone, ethylcellulose, methylcellulose, gumarabic, hydroxypropylcellulose, hydroxypropylmethylcellulose, sodiumcarboxymethylcellulose, propylene glycol and poly(sodium acrylate).Lubricants include, but are not limited to, magnesium stearate, talc andcalcium stearate. Antioxidants include, but are not limited to, ascorbicacid, α-tocopherol, ethoxyquin, dibutyl hydroxytoluene and dibutylhydroxyanisole. A coloring agent, a flavoring agent and the like may beadded. In tablets, granules and powder, a film coating may be applied,if needed.

Species of the infection-causing microorganism against whichadministration of the inventive medicines for immunostimulation andinfection-protection and -treatment, for preventing of treating sepsis,for preventing or treating septic shock or for preventing or treatingtoxin shock of the present invention comprising reduced riboflavinand/or a reduced riboflavin derivative or pharmacologically acceptablesalt thereof may be effective are not particularly limited. In general,the microorganisms include bacteria, fungi, parasites or protozoa,viruses, mycoplasmas, rickettsias and chlamydias. Particularly, it isfound that the medicines of the present invention may be highlyeffective against bacteria, fungi and parasites or protozoa.

Bacteria against which medicines of the present invention may acteffectively include, for example, Escherichia coli including variouspathogenic coliforms; bacteria belonging to the genus Salmonella such asSalmonella typhimurium, S. typhi, S. paratyphi, S. enteritidis, S.cholerasuis, S. gallinarum, S. abortasequi, S. abortasovis or S. dublin;bacteria belonging to the genus Shigella such as Shigella sonnei, S.dysenteriae, S. flexneri or S. boydii; bacteria belonging to the genusYersinia such as Yersinia pestis, Y. pseudotuberculosis or Y.enterocolitica; bacteria belonging to the genus Citrobacter such asCitrobacter freundii; bacteria belonging the genus Haemophilus such asHaemophilus somunus or H. parasuis; bacterial belonging to the genusActinobacillus such as Actinobacillus lignieresii; bacteria belonging tothe genus Pseudomonas such as Pseudomonas aeruginosa, P. mallei or P.fluorescens; bacteria belonging to the genus Bordetella such asBordetella bronchiseptica or B. pertussis; bacteria belonging to thegenus Brucella such as Brucella abortus, B. melitensis or B. canis;Neisseria meningitidis, N. gonorrhoeae; bacteria belonging to the genusBacterodes; bacteria belonging to the genus Fusobacterium; bacteriabelonging to the genus Veillonella; bacteria belonging to the genusCampylobacter such as Campylobacter fetus, C. sputorum, C. feacalis orC. jejuni; bacteria belonging to the genus Treponema; bacteria belongingto the genus Borrelia; bacteria belonging to the genus Spirichaeta;bacteria belonging to the genus Leptospira; bacteria belonging to thegenus Staphylococcus such as Staphylococcus aureus or S. epidermidis;bacteria belonging to the genus Streptococcus such as Streptococcuspyogens, S. pneumoniae or S. mutanns; bacteria belonging to the genusEnterococcus such as Enterococcus faecalis; bacteria belonging to thegenus Lactococcus such as Lactococcus garvidae; bacteria belonging tothe genus Bacillus such as Bacillus anthracis or B. cereus; bacteriabelonging to the genus Clostridium such as Clostridium perfingens, C.chauvoei, C. botulinum, C. tetani or C. septicum; bacteria belonging tothe genus Listeria such as Listeria monocytogenes; bacteria belonging tothe genus Erysipelothrix such as Erysipelothrix rhusiopathiae; bacteriabelonging to the genus Corynebacterium such as Corynebacterium renale,C. cystitidis, C. pseudotuberculosis or C. diphtheriae; bacteriabelonging to the genus Mycobacterium such as Mycobacterium tuberculosis,M. bovis, M. kansasii, M. ulcerance, M. goldnae, M. intracellulae, M.avium or M. leprae; bacteria belonging to the genus Actinomyces;bacteria belonging to the genus Serratia such as Serratia marcescens orS. rubidaea; bacteria belonging to the genus Vibrio such as Vibriocholerae or V. parahaemolyticus; bacteria belonging to the genusPasteurella such as Pasteurella multocida or P. haemolytica; bacteriabelonging to the genus Enterobactor such as Enterobactor cloacae;bacteria belonging to the genus Citrobacter such as Citrobacterfreundii; bacteria belonging to the genus Enterococcus such asEnterococcus seriolicida (including macrolide-resistant bacteria); andbacteria belonging to the genus Proteus. However, the present inventionis not intended to be limited by the species of the bacteria.

The present invention may also be effective against various resistantbacteria such as methicillin-resistant Staphylococcus aureus (MRSA) orpenicillin-resistant Streptococcus pneumoniae (PRSP).

Fungi against which medicines of the present invention may acteffectively include, for example, fungi belonging to the genusAspergillus such as Aspergillus fumigatus, A. flavus, A. terreus or A.niger; fingi belonging to the genus Candida such as Candida albicans, C.tropicalis, C. kurusei or C. pseudotropicalis; fingi belonging to thegenus Histoplasma such as Histoplasma capusulatum or H. fraciminosum;fingi belonging to the genus Microsporum such as Microsporum canis, M.distortum or M. nanam; fungi belonging to the genus Tricophyton such asTricophyton gallinae, T. rubrum or T. equinum; fingi belonging to thegenus Coccidioides such as Coccidioides immitis; fungi belonging to thegenus Blastomyces such as Blastomyces dermatitis; and fungi belonging tothe genus Cryptococcus such as Cryptococcus neoformans. However, thepresent invention is not intended to be limited by the species of thefungi.

Parasites against which the medicines of the present invention may acteffectively include, for example, malaria parasites such as Plasmodiummalariae, P. vivax, P. ovale, P. falciparum, P. knowlesi, P. cynomolgi,P. brasilianum or P. gallinaceum. In addition, also included areHistomonas tyzzer, Trypanosoma evavsi, T. hippicum, T. brucei, T.gambiense, T. cruzi, Leishmania donovani, L. tropoca, Trichomonasgallinae, T. gallinarum, T. hominis, T. foetus, T. vaginalis, Giardiaintestinalis, G. duodenalis, G. canis, Entamoeba histolytica, Eimeriatenella, E. necatrix, E. maxima, E. acervulina, E. bruneetti, E.meleagrimitis, E. adenoeides, E. zurnii, E. ellipsoidalis, E. bovis, E.arloingi, E. parva, E. debliecki, E. spinosa, E. stiedae, E. perforans,E. magna, E. mustelae, E. vison, Isospora bigemina, I. felis, I.ribolta, Klossiella muris, Hepatocystes kochi, Haemoproteus columbae,Leucocytozoon simondi, L. caulleryi, Babesia bigemina, B. gibsoni, B.canis, B. caballi, B. equi, Theileria parva, T. annulata, T. buffeli, T.sergenti, Anaplasma marginale, Toxoplasma gondii, Encephalitozoonlevaditi, Eperythrozoon wenyoni, Sarcocystis lindemanni, S. tenella,Plagiorchis muris, Dicrocoelium dendriticum, Eurytrema pancreaticum,Fasciola hepatica, F. gigantica, F. indica, Faciolopsis buski,Metrorchis orienntalis, M. akbidus, Microtrema truncatum, Centrocestusarmatus, Echinostoma revolutum, Schistosoma japonicum, S. haematobium,S. mansoni, Diphyllobothunri latum, D. erinacei, Diplogonoporus grandis,Anoplocephala perforiata, A. magna, Bertiella studeri, B. mucronata,Helicometra giardi, Dipylidium canium, hymenolepsis nana, H. exigua,Raillietina cesticillus, R. kashiwarensis, Taenia solium, T. pisiformis,Taeniarhynchus saginatus, Multiceps multiceps, M. serialis, Echinococcusgranulosus, E. multiocularis, Trichuris vulpis, T. suis, T. trichiura,Trichinella spiralis, Dioctophyma renale, Enterobius vermicularis,Ascaris lumbricoides, A. columnaris, Neoascaris vitulorum, Parascrisequorum, Toxocara canis, T. cati, Anisakis genus, Strongylus equinus, S.edentatus, S. vulgaris, Anchlostoma caninium, A. tubaeforme, A.duodenale, Necator americanus, Burgia malayi, Dirofilaria immitis, D.aculiuscula, Setaria equina, S. digitata, S. servi and S. marshalli.However, the present invention is not intended to be limited by thespecies of the parasites.

Viruses against which the medicines of the present invention may acteffectively include, for example, viruses belonging to the pox virusfamily such as vaccinia virus, variola virus, monkeypox virus, yabamonkey tumor virus, cowpox virus, ectromelia virus, contagious pustulardermatitis virus, bovine papular stomatitis virus, fowlpox virus orhuman verruca virus; viruses belonging to the herpes virus family suchas herpes simplex virus, variccela-zoster virus, human cytomegalovirus,malignant catarrhal fever virus, simian B virus, EB virus, pseudorabiesvirus, infectious bovine rhinotrancheitis virus, infectious avianLaryngotracheitis virus, Marek's disease virus, canine herpes virus,feline herpes virus, swine inclusion body rhinitis virus, carp pox virusor oncorhynchus masou virus; viruses belonging to the hepadonavirusfamily such as hepatitis B virus; viruses belonging to the adenovirusfamily such as human adenovirus, bovine adenovirus or simian adenovirus;viruses belonging to the papovavirus family such as human papillomavirus or JC virus; viruses belonging to the parvovirus family such asAleutian mink disease virus, HB virus, feline panluekopenia virus orcanine parvovirus; viruses belonging to the reovirus family such asIbaraki virus, bluetongue virus, Colorado mite fever virus or rotavirus; viruses belonging to the birnavirus family such as infectiousbursal disease virus or infectious pancreatic nucreosis virus; virusesbelonging to the orthomyxovirus family such as influenza A virus, fowlplaque virus or swine influenza virus; viruses belonging to theparamyxovirus family such as epidemic parotitis virus, mumpus virus,measles virus, Newcastle disease virus, parainfluenza virus, caninedistemper virus or RS virus; viruses belonging to the rhabdovirus familysuch as rabies virus, vesicular stomatitis virus, bovine ephemeral fevervirus, infectious hematopoietic necrosis virus or olivaceus (hirame)rhabdovirus; viruses belonging to the filovirus family such as Marburgvirus or Ebola virus; viruses belonging to the coronavirus family suchas human cold coronavirus, murine hepatitis virus, swine transmissiblegastroenteritis virus, fowl infectious bronchitis virus, felineinfectious peritonitis virus, equine arteritis virus or porcinereproductive respiratory syndrome virus; viruses belonging to thebunyavirus family such as hemorrhagic fever with renal syndrome virus,Akabane disease virus, Rift valley fever virus or Californiaencephalitis virus; viruses belonging to the togavirus family such asequine encephalitis virus, Murray valley encephalitis virus, measlesvirus or equine arteritis virus; viruses belonging to the flavivirusfamily such as yellow fever virus, Japanese encephalitis virus ordenguevirus; viruses belonging to the bunyamwera family such as RiftValley fever virus or Akabane virus; viruses belonging to the retrovirusfamily such as avian leukemia virus, avian eticuloendotheliosis virus,feline leukemia virus, equine infectious anaemia virus, humanspumavirus, human T-cell lymphotropic virus, human immunodeficiencyvirus or feline aquired immunodeficiency virus; viruses belonging to theiridovirus family such as lymphocele virus or sea beam iridovirusdisease virus; viruses belonging to the picornavirus family such ashuman coxsakie virus, human echo virus, human polio virus, rhinovirus orfoot-and-mouse disease virus; African swine fever virus; boma diseasevirus; and astrovirus. However, the present invention is not intended tobe limited by the species of the viruses.

Medicines of the present invention may also act effectively againstdiseases caused by proteins such as bovine spongiform encephalopathy,transmissible mink spongiform encephalopathy, scrapie orCreutzfeldt-Jacob disease.

Mycoplasmas against which the medicines of the present invention may acteffectively include, for example, mycoplasmas belonging to the genusMycoplasma, such as Mycoplasma mycoides, M. agalactiae, M.hyopneumoniae, M. capricolum, M. pulmonis or M. gallisepicum. However,the present invention is not intended to be limited by them.

Rickettsias against which the medicines of the present invention may acteffectively include, for example, rickettsias belonging to the generaRickettsia, Orientia, Coxiella, Ehrlichia, Wolbachia, Anaplasma,Haemobartonella, Eperythrozoon and Bartonella.

Chlamydia against which the medicines of the present invention may acteffectively include, for example, Chlamydia trachomatis, C. pneumoniae,C. psittaci or C. pecorum.

In the present invention, however, the microorganisms are not limited tothose mentioned above.

Medicines according to the present invention which comprise reducedriboflavin and/or a reduced riboflavin derivative or pharmacologicallyacceptable salt thereof as active ingredient may have a potentimmunomodulating effect.

The diseases on which administration of medicines according to thepresent invention comprising reduced riboflavin and/or a reducedriboflavin derivative or pharmacologically acceptable salt thereof asactive ingredient may be effective include infectious diseases by themicroorganisms mentioned above. The inventive medicines may haveexcellent effects on the infectious diseases as an immunostimulator andinfection-protective/treating medicine. The medicine may also be highlyeffective for preventing or treating malignant tumors, hematologicaldisorders, hepatic. diseases, collagen disease, renal metabolic diseasesand infections before and after operation.

The inventive medicines may also be effective on systemic inflammatoryresponse syndrome (SIRS) caused by the systemic microbial infection andhas a remarkable effect as a preventive/treating medicine for sepsis.Further, the inventive medicines may have extremely excellent effects asa preventive/treating medicine for septic shock and apreventive/treating medicine for toxin shock. They may also have aneffect as a preventive/treating medicines for disseminate intravascularcoagulation (DIC), multiple organ failure (MOF) and/or adult respiratorydistress syndrome (ARDS).

The inventive medicines may also have an excellent effect as apreventive/treating medicine for malarial diseases.

The inventive medicines may act. effectively on diseases. on whichimmunomodulating action is-effective, such as autoimmune diseases (e.g.,chronic rheumatoid arthritis, multiple sclerosis, amyotrophic lateralsclerosis, systemic lupus erythematosus, Behcet's disease, idiopathicthrombocytopenic purpura, myasthenia gravis, nodular arteriosclerosis,ulcerative colitis, Crohn's disease, atopic dermatitis and pollinosis),ischaemia-referfusion injuries (e.g., cerebral infarction and myocardialinfarction), hemorrhagic shock, photosensitive dermatitis, empyema,pyometra, otitis media, peritonitis, infectious endocarditis, diarrheaand dementia.

EXEMPLIFICATION

The present invention will be illustrated by the following examples, butis not intended to be limited by these examples.

Example 1 Effect on E. coli-Infected Model

Twelve mg of riboflavin sodium phosphate (FMN-Na, JP grade) wasdissolved in 40 ml of physiological saline and then added and mixed with480 mg of sodium hydrosulfite to produce leucoflavin phosphate (FMN-H₂).

Male mice (Slc: ICR, 8-week old) (10 mice per each test group) wasadministered intraperitoneally with each of riboflavin sodium phosphate(dosage: 12.5, 25, 50 and 100 mg/kg in dosage volumes of 0.125, 0.25,0.5 and 1 ml, respectively) and leucoflavin phosphate (dosage: 1.25,2.5, 5 and 10 mg/kg in dosage volumes of 0.125, 0.25, 0.5 and 1 ml,respectively) which had been dissolved in physiological saline andsterilized by filtration. Twenty-four hours after the administration, E.coli E01292 strain was inoculated subcutaneously at a dose of 5.3×10⁷CFU/mouse (suspended in 0.2 ml of physiological saline), and thesurvival rate was determined 4 days after the inoculation. In thecontrol group, 1.0 ml of physiological saline was administeredintraperitoneally instead of the drugs. The determination of themedicinal efficacy was conducted by χ² test. The results are shown inTable 1.

TABLE 1 Survival rate Test group survival number (survival rate %)Control 0 (0) VB₂Na 12.5 mg/kg 1 (10) VB₂Na 25 mg/kg 3 (30) VB₂Na 50mg/kg 5 (50)* VB₂Na 100 mg/kg 9 (90)** FMN-H₂ 1.25 mg/kg 0 (10) FMN-H₂2.5 mg/kg 4 (40) FMN-H₂ 5 mg/kg 6 (60)** FMN-H₂ 10 mg/kg 9 (90)** VB₂Na:riboflavin sodium phosphate FMN-H₂: leucoflavin phosphate *:P < 0.05,**:P < 0.01

As shown in Table 1, leucoflavin phosphate showed about 10-fold greaterinfection-protective effect against E. coli compared with riboflavinsodium phosphate.

Example 2 Effect on Staphylocuccus aureus-Infected Model

Riboflavin sodium phosphate (FMN-Na, JP grade) and leucoflavin sodiumphosphate (FMN-H₂) prepared in Example 1 were used.

Male mice (Slc: ICR, 8-week old) (10 mice per each test group) wasadministered intraperitoneally with each of riboflavin sodium phosphate(dosage: 25, 50 and 100 mg/kg in dosage volumes of 0.25, 0.5 and 1 ml,respectively) and leucoflavin phosphate (dosage: 2.5, 5 and 10 mg/kg indosage volumes of 0.25, 0.5 and 1 ml, respectively) which had beendissolved in physiological saline and sterilized by filtration.Twenty-four hours after the administration, Staphylococcus aureus BO-72strain was administered intravenously at a dose of 8.7×10⁷ CFU/mouse(suspended in 0.2 ml of physiological saline), and the survival rate wasdetermined 10 days after the inoculation. In the control group, 1.0 mlof physiological saline was administered intraperitoneally instead ofthe drugs. The determination of the medicinal efficacy was conducted byχ² test. The results are shown in Table 2.

TABLE 2 Survival rate Test group survival number (survival rate %)Control 0 (0) VB₂Na 25 mg/kg 2 (20) VB₂Na 50 mg/kg 4 (40) VB₂Na 100mg/kg 7 (70)** FMN-H₂ 2.5 mg/kg 3 (30) FMN-H₂ 5 mg/kg 6 (60)** FMN-H₂ 10mg/kg 8 (80)** VB₂Na: riboflavin sodium phosphate FMN-H₂: leucoflavinphosphate *:P < 0.05, **:P < 0.01

As shown in Table 2, leucoflavin phosphate showed about 10-fold greaterinfection-protective effect against Staphylococcus aureus compared withriboflavin sodium phosphate.

Example 3 Effect on Endotoxin Shock Model

Riboflavin sodium phosphate (FMN-Na, JP grade) and leucoflavin sodiumphosphate (FMN-H₂) prepared in Example 1 were used.

Male mice (Slc: ICR, 6-week old) (10 mice per each test group) wasadministered intraperitoneally with lipopolysaccharide from E. coliserum type 0111-B4 (Sigma; abbreviated as “LPS” hereinbelow) at a doseof 15 mg/kg.

Six hours after the administration, each of riboflavin sodium phosphate(dosage: 5, and 20 mg/kg in dosage volumes of 0.25, 0.5 and 1 ml,respectively) and leucoflavin phosphate (dosage: 2.5, 5 and 10 mg/kg indosage volumes of 0.25, 0.5 and 1 ml, respectively) which had beendissolved in physiological saline and sterilized by filtration wasadministered intraperitoneally. The survival rate was determined 4 daysafter the administration. In the control group, 1.0 ml of physiologicalsaline was administered intraperitoneally instead of the drugs. Thedetermination of the medicinal efficacy was conducted by χ² test. Theresults are shown in Table 3.

TABLE 3 Survival rate Test group survival number (survival rate %)Control 0 (0) VB₂Na 5 mg/kg 3 (30) VB₂Na 10 mg/kg 4 (40) VB₂Na 20 mg/kg7 (70)** FMN-H₂ 2.5 mg/kg 2 (20) FMN-H₂ 5 mg/kg 6 (60)** FMN-H₂ 10 mg/kg8 (80)** VB₂Na: riboflavin sodium phosphate FMN-H₂: leucoflavinphosphate *:P < 0.05, **:P < 0.01

As shown in Table 3, leucoflavin phosphate showed about 2-fold greaterlife-saving effect on endotoxin shock than riboflavin sodium phosphate.

1. A method for treating sepsis, comprising administering a pharmacologically effective amount of a reduced riboflavin and/or a reduced riboflavin derivative or pharmacologically acceptable salt thereof to a subject in need thereof, wherein the reduced riboflavin is selected from the group consisting of leucoflavin and pharmaceutically acceptable salts thereof, and wherein the reduced riboflavin derivative is selected from the group consisting of leucoflavin mononucleotide, leucoflavin adenine dinucleotide and pharmaceutically acceptable salts thereof.
 2. The method according to claim 1, wherein the pharmaceutically acceptable salt of leucoflavin is leucoflavin phosphate ester. 